AcuteandchronicQfeverinfectionsinhumansarecausedbyCoxiellaburnetii,whichtrafficsto and replicates in Coxiella-containing vacuoles (CCV). Infection suppresses macrophage activation in a type 4 secretion system-dependent manner, hence our central hypothesis is characterizationofT4SS-dependenteffectormoleculesinvitroandinvivowilldefine?stealthy? virulence genes. This application will identify T4SS-dependent virulence determinants that modulate macrophage signaling pathways using pathogen effector mutants and host signaling mutants that are essential for restricting replication or modulating response to infection. Aim 1. IdentifytherangeofT4SSdependentmanipulationofmacrophage.Theworkinghypothesis ismultipleT4SSeffectorsmanipulatetheresponsetoinfectioninmacrophages.Usingaprimary bonemarrowderivedmacrophage(BMDM)cellculturethatenablesC.burnetii,RSA439(NMII) to replicate. We will extensively characterize BMDM using transcriptomic (including single cell analysis),metabolomicsandflowcytometricanalysistocomprehensivelymapT4SSdependent pathway modulation of macrophage activation. Aim 2. Identify T4SS effectors which manipulate immune signaling. The working hypothesis is T4SS effectors target specific activation signaling pathways. To identify this broad class of T4SS effectors, tagged C. burnetii T4SS substrates will be transfected into macrophages in the context of signaling agonists. A complementary approach will use T4SS mutants to identify infection that does not modulate innateactivation.Aim3.EstablishmechanisticbasisforT4SSeffectormodulation.Current candidatesderivedfrompreliminarydatainclude;?a)2Ankyrinrepeat-containingproteinswhich dampenagonistdrivenNF-?b?;?b)5T4SSeffectorsthatareessentialforreplicationinBMDM;?and c) 3 T4SS effectors which traffic to the nucleus (nucleomodulins). We will identify host binding partners using pull-down methods and define their role in disease using host knock-down or knock-outapproaches.EacheffectorwillbeanalyzedwitheitherTnorsite-specificmutants.